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Biological evaluation of biomaterials, alkaloids from Pilocarpus microphyllus, in human neutrophils: toxicity and anti-inflammatory activity

2016; Frontiers Media; Volume: 4; Linguagem: Inglês

10.3389/conf.fbioe.2016.01.00155

2296-4185

Magalhaes Rocha Talita, Luzia Kalyne Almeida Moreira Leal, Fernandes Lima David, Roberto Leite Jos�, Socorro Barros Viana Glauce, Dislich Ropke Cristina,

Biological Research and Disease Studies

Event Abstract Back to Event Biological evaluation of biomaterials, alkaloids from Pilocarpus microphyllus, in human neutrophils: toxicity and anti-inflammatory activity Talita Magalhaes Rocha1, 2, Luzia Kalyne Almeida Moreira Leal1, 2, David Fernandes Lima3, José Roberto Leite4, Glauce Socorro Barros Viana1 and Cristina Dislich Ropke5 1 Federal University of Ceará, Department of Physiology and Pharmacology, Brazil 2 Federal University of Ceará, Department of Pharmacy, Brazil 3 Federal University of Vale of the São Francisco, Brazil 4 Federal University of Piauí, Brazil 5 Phytobios, Brazil Introduction: Pilocarpus microphyllus, popularly known as jaborandi, is a small tree extensively used by Brazilian industries and rich in imidazole alkaloids, such as pilocarpine, epiisopilosine (EPIL) and epiisopiloturine (EPIT). Objective: The aim of the present study was to evaluate the toxicity, anti-inflammatory and antioxidant activities of the EPIL and EPIT in human neutrophils. Methods: The EPIL (≥ 90% Purity) and EPIT (99,7% purity) were obtained from a residual product obtained during the isolation process of pilocarpine from plant leaves. Polymorphonuclear cells - PMNs (2.5 x 106cells/mL), predominantly neutrophils (90%) with cell viability of 95% (Tripan blue assay) were isolated from human blood residual product[1]. The toxicity of alkaloids (10, 50, 100 μg/mL) was evaluated by MTT test (620nm)[2] and lactate dehydrogenase (LDH) activity (340 nm)[3]. The potential of alkaloids in modulating pro-inflammatory mechanisms of human neutrophils was evaluated by two assays, degranulation and burst respiratory assays. The cells were incubated with alkaloids (1, 10, 25, 50, 100 μg/mL), indomethacin (INDO-standard drug, 36µg/mL), DMSO 1% (vehicle/control) or HBSS (sham), and then the cells were activated by addition of PMA (0,1 µM) with consequent release of myeloperoxidase (MPO) which was measured at 450nm[4],[5]. The antioxidant activity was determined by chemiluminescence (QL) assay[6]. The PMNs (5x106 cells/mL) were incubated (37° C) with EPIT or EPIL (1 - 100 μg/mL), HBSS (sham), quercetin (Querc, 25μg/mL, standard drug) or DMSO 1% (vehicle/control), plus probe luminol (lum, 280 μM). After stimulation with PMA (0,1 μM), the production of QL for 20 min was registered. The results were expressed as a mean ± standard error, and analyzed by ANOVA (followed by Tukey test, p < 0.05). Results and Discussion: The addition of EPIT or EPIL up to the highest concentration (100 µg/mL) in human neutrophils suspension did not reduce significantly the cells viability evaluated by LDH activity or MTT test. The maximal reduction of neutrophil degranulation induced by EPIT was approximately 35%, whereas EPIL inhibited by 68 %, an effect comparable to INDO, standard drug (inhibition: 62 %). Both alkaloids reduced ROS production measured as QL by lum in human neutrophils. However, EPIL (inhibition: 74 %) again was much more potent than EPIT (inhibition: 33 %), showing an effect similar to standard drug quercetin (75 %). Conclusions: The alkaloids from Pilocarpus microphyllus modulated pro-inflammatory mechanisms of human neutrophils through the MPO-H2O2-HOCl system and this effect does not seem to be related to a cytotoxic action. EPIL showed a higher anti-inflammatory effect than EPIT, and additional studies are needed to establish the precise mechanism of action of this molecule. CNPq; CAPESReferences:[1] LUCISANO, Y. M.; MANTOVANI, B. Lysossomal enxime release from polymorfonuclear leukocytes induced by immune complexes of IgM and IgG. The Journal of Immunology. v. 132, p. 2015-2020, 1984.[2] MOSMANN, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods. v. 65, p. 55-63, 1983.[3] BERGMEYER HU, BERNT E. Lactate dehydrogenase. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis. London: Academic Press; 1963. pp. 574–579.[4] ÚBEDA, A.; FERRÁNDIZ, M. L.; HERENCIA, F. Activación celular: desgranulación leucocitária. In: ALCARAZ, M. J.; CALIXTO, J. B.; DELGADO, R. Técnicas in vitro para el studio de fármacos antiinflamatório. CYTED – Subprograma X Projecto X.6. Espanha, 2002.[5] DE YOUNG, L.M., KHEIFETS, J.B., BALLARON, S.J., YOUNG, J.M. Edema and cell infiltration in the phorbol ester-treated mouse ear are temporally separate and can be differentially modulated by pharmacologic agents. Agents and Actions, v. 26, p.335- 341, 1989.[6] KUDOH, S.; KATSUHIKO, S.; YAMADA, M.; LIU, Q.; NAKAJI, S.; SUGAWARA, K. Contribution of nitric oxide syntase to human neutrophil chemoluminescence. Luminescence. v. 14, p. 335-39, 1999. Keywords: in vitro, cell, stimuli-response, Cell modulation Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Role of biomaterials in inflammation Citation: Magalhaes Rocha T, Kalyne Almeida Moreira Leal L, Fernandes Lima D, Roberto Leite J, Socorro Barros Viana G and Dislich Ropke C (2016). Biological evaluation of biomaterials, alkaloids from Pilocarpus microphyllus, in human neutrophils: toxicity and anti-inflammatory activity. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00155 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Talita Magalhaes Rocha Luzia Kalyne Almeida Moreira Leal David Fernandes Lima José Roberto Leite Glauce Socorro Barros Viana Cristina Dislich Ropke Google Talita Magalhaes Rocha Luzia Kalyne Almeida Moreira Leal David Fernandes Lima José Roberto Leite Glauce Socorro Barros Viana Cristina Dislich Ropke Google Scholar Talita Magalhaes Rocha Luzia Kalyne Almeida Moreira Leal David Fernandes Lima José Roberto Leite Glauce Socorro Barros Viana Cristina Dislich Ropke PubMed Talita Magalhaes Rocha Luzia Kalyne Almeida Moreira Leal David Fernandes Lima José Roberto Leite Glauce Socorro Barros Viana Cristina Dislich Ropke Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.